This project is directed at understanding the mechanisms of HIV-1 binding, entry, replication and assembly, particularly in macrophages, and in developing an effective strategy to prevent and/or inhibit infection. Immunodeficiency, the consequence of HIV-1 infection, predisposes the host to opportunistic infections. In turn, opportunistic infections influence target cell susceptibility to HIV-1 infection and replication. Using M. avium as a model co-pathogen, we have defined multiple viral permissive factors. Moreover, immune activation as typically occurs in tonsils and non-infectious mucosal inflammatory lesions may also be associated with proximal sites of viral replication. These connections between activation/inflammation and enhancement of HIV-1 infection warrant further elucidation of the factors promoting permissiveness to HIV-1. Infection of human macrophages in an in vitro model revealed a pattern of signal transduction, expression of immediate early genes, and downstream genes associated with viral replication by cDNA microarray analyses. As the association between signaling cascades, gene transcription and macrophage-specific viral dynamics is elucidated, our goal is to identify new cellular targets to interfere with HIV replication. In this regard, our focus has been on the characterization of annexin II, p21 and APOBEC3 family members as host cell molecules either commandeered by HIV or as resistance factors, which can be exploited to disrupt the virus life cycle.[unreadable] [unreadable] Host Cell Factors as Targets to Disrupt the HIV Life Cycle:[unreadable] The complex life cycle of HIV involves critical functional interactions with host cell components, and these host cell factors may represent targets for therapeutic intervention. Furthermore, HIV drugs designed to interfere with requisite utilization of host cell factors by the virus are considered less likely to foster development of resistant strains than drugs directed against viral components prone to mutation. Cellular genes required for binding/entry and also productive infection by HIV have been identified as potential targets for anti-HIV drug development. Consequently, identification of cellular genes that are involved in early events, whether at the level of virion entry, viral DNA synthesis or proviral integration or production of viral transcripts remain a high priority as pivotal targets for blocking the viral life cycle. In studies to characterize the mechanism by which the innate host defense molecule, SLPI, interferes with macrophage infection by HIV, a membrane binding molecule, annexin II, was identified which may serve as a conduit during HIV entry and be a potential target for intervention. Downregulation of annexin II by using siRNA methodology was associated with an inhibitory effect on early events in HIV infection. The amount of viral DNA synthesis measured postinfection by nested PCR showed that an early step in the life cycle was halted by the absence of annexin II, and evidence indicates that inhibition occurred postbinding, likely at the level of fusion/entry, implicating annexin II as a targetable viral entry cofactor. [unreadable] [unreadable] Among the additional genes identified which are obligatory to the successful life cycle of HIV in macrophages is p21. HIV infection induced the transcriptional regulation of genes associated with host defense, signal transduction, apoptosis and cell cycle, of which p21, a cyclin-dependent kinase inhibitor 1A(CDKN1A) is the most prominent. Since the HIV viral protein R (Vpr) facilitates virus replication in non-dividing cells, and is required for efficient HIV-1 production in late stages of replication in tissue macrophages, we assessed the potential contribution of Vpr in mediating p21 induction. Our studies demonstrate that Vpr represents at least one mechanism by which HIV-1 drives p21 transcription. Inhibition of p21 reduced HIV-1 replication by siRNA, antisense and a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), known to influence the kinase inhibitor expression, suppressed viral replication. These data implicate this host cell molecule as a requisite macrophage facilitator of viral replication. Moreover, regulators of this molecule, such as CDDO, may provide an interventional approach to modulate HIV-1 replication. In addition to further delineation of the mechanistic actions of CDDO in macrophages and T cells, a clinical trial using CDDO as therapy in HIV positive individuals is under development.[unreadable] [unreadable] An additional host cell molecule(s) which is involved in the HIV life cycle is apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), which is an innate intracellular protein with lethal activity against HIV. Originally thought to be constitutively expressed and depleted by the virus, our new studies demonstrate that the levels of this host defense molecule can be augmented by interferon, concomitant with resistance to HIV. [unreadable] [unreadable] Mucosal targeting and resistance factors in HIV infection:[unreadable] The human tonsil represents a target and potential reservoir for HIV replication. Oral HIV-1 transmission, although rare, does not preclude this secondary lymphoid organ from becoming heavily infected either initially or secondarily as the disease progresses. In order to identify what unique factors favor the massive infection seen in tonsil lymphoid tissues, tonsil susceptibility to HIV infection was compared with that of peripheral blood mononuclear cells (PBMC) and resting T lymphocytes. In these studies, we identified several unique aspects of the micromilieu of the tonsil that may support the permissive nature of the tonsil to HIV infection. In culture, as occurs in situ, tonsil cells are more permissive for HIV, independent of additional exogenous stimulation. By a battery of analyses, including transcriptional, signal transduction, and proteomic, we identified multiple contributory elements to the retroviral permissive nature of the tonsil. Such a profile of immune regulation is necessary to control immune activation, yet maintain a state of readiness in an environment constantly bombarded with antigens and pathogens, but which unfortunately represents a favorable condition for HIV replication. One of these immunoregulatory molecules, suppressor of cytokine signaling (SOCS), which inhibits JAK-STAT and TLR signaling pathways, is elevated in the tonsil and may contribute to the blockade of an IFN signaling cascade to effectively disengage IFN antiviral activity. These and other unique tonsillar microenvironmental cues favor a milieu that renders tonsil cells susceptible to HIV infection and underscore the role of local immune regulatory mechanisms which may account for the selectivity of HIV infection within mucosal compartments. In ongoing related studies, the contribution of tonsil Treg, macrophages and dendritic cells as potential regulators and targets for the virus in HIV pathogenesis is also being dissected. Further understanding of these immunoregulatory pathways and their contribution to HIV pathogenesis will promote strategies for manipulating vulnerable mucosal compartments.